Epub: Condie et al. Two-Photon Fluorescent Imaging of Myelination in the Spinal Cord. ChemMedChem. 2012 Nov 7. doi: 10.1002/cmdc.201200343.Myelination is a fundamental biological process in the vertebrate nervous system. Damage to or malformation of myelin can lead to various neurological diseases; for example, demyelination in the spinal cord is a major cause of paralysis of patients suffering from multiple sclerosis and related diseases. The ability to directly track myelin levels in the spinal cord is needed in order to assess the efficacy of therapeutics in promoting myelin repair. To address this unmet need, 4-((E)-4-((E)-4-aminostyryl)-2,5-dimethoxystyryl)-N-methylaniline, known as Case Imaging Compound (CIC), has been developed as a myelin-targeted fluorescent imaging agent that selectively binds to myelin. CIC was synthesized via an improved route and evaluated as a fluorescent probe for two-photon fluorescent imaging of myelin in the spinal cord in both demyelinated and dysmyelinated models. In vitro and ex vivo tissue staining both suggest that CIC selectively binds to myelin in animal models. Further evaluation in animal models indicated that CIC is sensitive to differences in myelin content in healthy versus pathological myelin. CIC could potentially be useful in the development and evaluation of novel therapies for multiple sclerosis and other demyelinating diseases.

Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to a very high depth, that is up to about one millimeter. Being a special variant of the multiphoton fluorescence microscope, it uses red-shifted excitation light which can also excite fluorescent dyes. However for each excitation, two photons of the infrared light are absorbed. Using infrared light minimizes scattering in the tissue. Due to the multiphoton absorption the background signal is strongly suppressed. Both effects lead to an increased penetration depth for these microscopes. However, the resolution remains diffraction-limited. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection and reduced phototoxicity. The concept of two-photon excitation is based on the idea that two photons of comparably lower energy than needed for one photon excitation can also excite a fluorophore in one quantum event. Each photon carries approximately half the energy necessary to excite the molecule. An excitation results in the subsequent emission of a fluorescence photon, typically at a higher energy than either of the two the two excitatory photons.

Two photon microscopy allows imaging in living animals and this has revolutionised our knowledge as cells are not static as in standard microscopy but alive and moving. I have just seen videos of microglia sampling every bit of the CNS all the time. This study could help us watch myelination, remyelination as it happens. There are other studies doing this!